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Article in English | WPRIM | ID: wpr-878357


Objective@#The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma.@*Method@#A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma.@*Results@#Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log @*Conclusion@#The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.

DNA, Viral/analysis , Diagnostic Tests, Routine/methods , Dried Blood Spot Testing/methods , HIV Infections/diagnosis , HIV-1/isolation & purification , Hepacivirus/isolation & purification , Hepatitis C/diagnosis , RNA, Viral/analysis , Sensitivity and Specificity , Specimen Handling/methods , Syphilis/diagnosis , Treponema pallidum/isolation & purification
Article in English | WPRIM | ID: wpr-877687


INTRODUCTION@#Pregnant women are reported to be at increased risk of severe coronavirus disease 2019 (COVID-19) due to underlying immunosuppression during pregnancy. However, the clinical course of COVID-19 in pregnancy and risk of vertical and horizontal transmission remain relatively unknown. We aim to describe and evaluate outcomes in pregnant women with COVID-19 in Singapore.@*METHODS@#Prospective observational study of 16 pregnant patients admitted for COVID-19 to 4 tertiary hospitals in Singapore. Outcomes included severe disease, pregnancy loss, and vertical and horizontal transmission.@*RESULTS@#Of the 16 patients, 37.5%, 43.8% and 18.7% were infected in the first, second and third trimesters, respectively. Two gravidas aged ≥35 years (12.5%) developed severe pneumonia; one patient (body mass index 32.9kg/m2) required transfer to intensive care. The median duration of acute infection was 19 days; one patient remained reverse transcription polymerase chain reaction (RT-PCR) positive >11 weeks from diagnosis. There were no maternal mortalities. Five pregnancies produced term live-births while 2 spontaneous miscarriages occurred at 11 and 23 weeks. RT-PCR of breast milk and maternal and neonatal samples taken at birth were negative; placenta and cord histology showed non-specific inflammation; and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulins were elevated in paired maternal and umbilical cord blood (n=5).@*CONCLUSION@#The majority of COVID-19 infected pregnant women had mild disease and only 2 women with risk factors (obesity, older age) had severe infection; this represents a slightly higher incidence than observed in age-matched non-pregnant women. Among the women who delivered, there was no definitive evidence of mother-to-child transmission via breast milk or placenta.

Abortion, Spontaneous/epidemiology , Adult , COVID-19/transmission , COVID-19 Nucleic Acid Testing , COVID-19 Serological Testing , Cohort Studies , Disease Transmission, Infectious/statistics & numerical data , Female , Fetal Blood/immunology , Humans , Infectious Disease Transmission, Vertical/statistics & numerical data , Live Birth/epidemiology , Maternal Age , Milk, Human/virology , Obesity, Maternal/epidemiology , Placenta/pathology , Pregnancy , Pregnancy Complications, Infectious/physiopathology , Pregnancy Outcome/epidemiology , Pregnancy Trimester, First , Pregnancy Trimester, Second , Prospective Studies , RNA, Viral/analysis , Risk Factors , SARS-CoV-2 , Severity of Illness Index , Singapore/epidemiology , Umbilical Cord/pathology , Young Adult
Article in Spanish | LILACS | ID: biblio-1292488


INTRODUCCIÓN: En 6 meses se notificaron más de 400 mil fallecidos por COVID-19. Han surgido múltiples investigaciones para comprender su etiopatogenia, siendo la autopsia médica uno de los mejores procedimientos para obtener información. Presentamos una revisión respecto a hallazgos post mortem publicados hasta mayo, 2020. RESULTADOS: Se recolectaron 12 estudios, de un total de 109 pacientes cuyo deceso fue por complicación respiratoria, predominó el sexo masculino, edad avanzada y con múltiples comorbilidades. El estudio PCR se realizó principalmente para diagnóstico. Se demostró ARN viral en riñón, hígado, corazón, cerebro y otros órganos. Los autores relataron presencia de micro y/o macro trombosis, en 50 de 109 casos, sobre todo a nivel pulmonar y renal, de tipo microscópica y relacionados a signos de shock. Desde la perspectiva anatomopatológica, se centra en alteraciones pulmonares y renales: daño alveolar difuso, injuria tubular aguda, microtrombos y otros signos de alteración microcirculatoria. Los estudios inmunohistoquímicos, de inmunofluoresencia y microscopía electrónica sugieren tropismo del virus por células epiteliales y estromales a nivel pulmonar y renal. En otros órganos se encuentran elementos morfológicos inespecíficos, atribuibles a patologías de base o shock. CONCLUSIÓN: El patrón histopatológico de daño alveolar difuso es frecuente, principalmente en fase exudativa o temprana. En el tejido renal destaca la injuria tubular aguda y daño microcirculatorio. El número y la descripción de muestras en otros órganos es reducida, siendo necesaria mayor casuística. La trombosis, es un trastorno prevalente en pulmones y riñones de pacientes con signos de shock. El tipo de trombo con más frecuencia descrito, es el microtrombo. Si bien se puede explicar como gatillante del fenómeno trombótico la interacción entre agente y huésped, otros factores deben ser estudiados para dilucidar la patogenia.

Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Thrombosis/pathology , COVID-19/pathology , Autopsy , Thrombosis/diagnosis , RNA, Viral/analysis , Polymerase Chain Reaction , COVID-19/diagnosis , COVID-19/mortality , Kidney/pathology , Kidney/virology , Liver/pathology , Liver/virology , Lung/pathology , Lung/virology
Braz. j. med. biol. res ; 52(11): e8339, 2019. tab, graf
Article in English | LILACS | ID: biblio-1039262


A progressive increase in the circulation of arboviruses in tropical countries has been observed, accounting for 700,000 yearly deaths in the world. The main objective of this article was to identify the presence of Zika (ZIKV), dengue (DENV), and Chikungunya (CHIKV) viruses in immature stages of Aedes aegypti and Ae. albopictus. Household collections of immature phases of the vectors were carried out in the years 2015 and 2016. A total of 2902 dwellings were visited and the rate of infestation with larvae and pupae of Aedes mosquitoes was 283/1462 (19.4%) in March 2015 and 55/1440 (3.8%) in June 2015. In March 2015, 907 larvae/pupae were collected (583 or 64.3% of Ae. aegypti and 324 or 35.7% of Ae. albopictus) while in June 2015 there was a reduction in the number of immature forms found: 197 larvae/pupae (121 or 61.4% of Ae. aegypti and 76 or 38.6% of Ae. albopictus). This reduction was accompanied by a decrease in suspected human ZIKV cases from March to June 2015. The RT-qPCR performed in 18 pools identified that three (two of Ae. aegypti and one of Ae. albopictus) were positive for ZIKV, and none were positive for DENV or CHIKV. Our findings demonstrated that ZIKV was present in immature stages of insect vectors in the study region at least five months prior to the peak of ZIKV associated cases. Xenomonitoring of immature phases of the vectors may prove useful for predicting outbreaks.

Humans , Animals , Chikungunya virus/isolation & purification , Aedes/virology , Dengue Virus/isolation & purification , Zika Virus/isolation & purification , Mosquito Vectors/virology , Seasons , RNA, Viral/analysis , Aedes/classification , Real-Time Polymerase Chain Reaction , Zika Virus Infection/transmission , Mosquito Vectors/classification
Rev. Soc. Bras. Med. Trop ; 50(4): 465-469, July-Aug. 2017. tab, graf
Article in English | LILACS | ID: biblio-896994


Abstract INTRODUCTION: Chikungunya fever is a condition resulting from infection by chikungunya virus (CHIKV), an Aedes sp.-transmitted virus. This disease has been diagnosed in thousands of cases in the Americas, particularly in Brazil, in recent years, and there is an ongoing epidemic of chikungunya fever in Brazil that began in 2014. Clinical diagnosis is difficult; only a few cases have been confirmed by laboratory tests due to the low number of specific, efficient tests available for virus or antibody detection. Here, we aimed to evaluate different polymerase chain reaction (PCR) approaches for detection of CHIKV genetic material. METHODS: Specific primers and probes within the viral capsid gene region were designed for this work. To evaluate the analytic sensitivity of detection, human sera were spiked with serial dilutions of the viral stock. Several PCR protocols were performed to investigate the sensitivity of CHIKV RNA detection in serum dilutions ranging from 106 to 1 PFU equivalents. RESULTS: The technique showing the greatest sensitivity was a real-time PCR assay using specific probes that could detect the genetic material of the virus at all dilutions, followed by conventional PCR. Digital PCR showed low sensitivity and was much more expensive than other technologies. Digital PCR should be used for specific purposes other than clinical diagnosis. CONCLUSIONS: Although quantitative PCR using probes was more expensive than the use of intercalating dyes or conventional PCR, it had the highest sensitivity out of all tested PCR approaches.

Humans , RNA, Viral/analysis , Chikungunya virus/genetics , DNA Primers/genetics , Chikungunya Fever/diagnosis , Sensitivity and Specificity , Real-Time Polymerase Chain Reaction
Braz. j. microbiol ; 48(2): 373-379, April.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-839368


Abstract Hepatitis E virus is responsible for acute and chronic liver infections worldwide. Swine hepatitis E virus has been isolated in Brazil, and a probable zoonotic transmission has been described, although data are still scarce. The aim of this study was to investigate the frequency of hepatitis E virus infection in pigs from a small-scale farm in the rural area of Paraná State, South Brazil. Fecal samples were collected from 170 pigs and screened for hepatitis E virus RNA using a duplex real-time RT-PCR targeting a highly conserved 70 nt long sequence within overlapping parts of ORF2 and ORF3 as well as a 113 nt sequence of ORF2. Positive samples with high viral loads were subjected to direct sequencing and phylogenetic analysis. hepatitis E virus RNA was detected in 34 (20.0%) of the 170 pigs following positive results in at least one set of screening real-time RT-PCR primers and probes. The swine hepatitis E virus strains clustered with the genotype hepatitis E virus-3b reference sequences in the phylogenetic analysis and showed close similarity to human hepatitis E virus isolates previously reported in Brazil.

Animals , Swine Diseases/epidemiology , Hepatitis E virus/isolation & purification , Hepatitis E virus/classification , Hepatitis E/veterinary , Phylogeny , Swine , Swine Diseases/virology , Brazil , RNA, Viral/analysis , RNA, Viral/genetics , Cluster Analysis , Prevalence , Hepatitis E virus/genetics , Hepatitis E/epidemiology , Hepatitis E/virology , Sequence Homology , Sequence Analysis, DNA , Feces/virology , Real-Time Polymerase Chain Reaction
Mem. Inst. Oswaldo Cruz ; 111(5): 287-293, May 2016. graf
Article in English | LILACS | ID: lil-782050


An unusually high incidence of microcephaly in newborns has recently been observed in Brazil. There is a temporal association between the increase in cases of microcephaly and the Zika virus (ZIKV) epidemic. Viral RNA has been detected in amniotic fluid samples, placental tissues and newborn and fetal brain tissues. However, much remains to be determined concerning the association between ZIKV infection and fetal malformations. In this study, we provide evidence of the transplacental transmission of ZIKV through the detection of viral proteins and viral RNA in placental tissue samples from expectant mothers infected at different stages of gestation. We observed chronic placentitis (TORCH type) with viral protein detection by immunohistochemistry in Hofbauer cells and some histiocytes in the intervillous spaces. We also demonstrated the neurotropism of the virus via the detection of viral proteins in glial cells and in some endothelial cells and the observation of scattered foci of microcalcifications in the brain tissues. Lesions were mainly located in the white matter. ZIKV RNA was also detected in these tissues by real-time-polymerase chain reaction. We believe that these findings will contribute to the body of knowledge of the mechanisms of ZIKV transmission, interactions between the virus and host cells and viral tropism.

Humans , Male , Female , Infant, Newborn , Adult , Infectious Disease Transmission, Vertical , Microcephaly/virology , Viral Tropism/physiology , Zika Virus Infection/congenital , Zika Virus/physiology , Amniotic Fluid/virology , Brain/embryology , Brain/virology , Immunohistochemistry , Infant, Newborn , Placenta/virology , Pregnancy , RNA, Viral/analysis
Article in English | WPRIM | ID: wpr-151582


BACKGROUND: It is crucial to understand the current status of clinical laboratory practices for the largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infections in the Republic of Korea to be well prepared for future emerging infectious diseases. METHODS: We conducted a survey of 49 clinical laboratories in medical institutions and referral medical laboratories. A short questionnaire to survey clinical laboratory practices relating to MERS-CoV diagnostic testing was sent by email to the directors and clinical pathologists in charge of the clinical laboratories performing MERS-CoV testing. The survey focused on testing volume, reporting of results, resources, and laboratory safety. RESULTS: A total of 40 clinical laboratories responded to the survey. A total of 27,009 MERS-CoV real-time reverse transcription PCR (rRT-PCR) tests were performed. Most of the specimens were sputum (73.5%). The median turnaround time (TAT) was 5.29 hr (first and third quartile, 4.11 and 7.48 hr) in 26 medical institutions. The median TAT of more than a half of the laboratories (57.7%) was less than 6 hr. Many laboratories were able to perform tests throughout the whole week. Laboratory biosafety preparedness included class II biosafety cabinets (100%); separated pre-PCR, PCR, and post-PCR rooms (88.6%); negative pressure pretreatment rooms (48.6%); and negative pressure sputum collection rooms (20.0%). CONCLUSIONS: Clinical laboratories were able to quickly expand their diagnostic capacity in response to the 2015 MERS-CoV outbreak. Our results show that clinical laboratories play an important role in the maintenance and enhancement of laboratory response in preparation for future emerging infections.

Clinical Laboratory Services/standards , Clinical Laboratory Techniques/instrumentation , Coronavirus Infections/diagnosis , Disease Outbreaks , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction , Republic of Korea/epidemiology , Sputum/virology , Surveys and Questionnaires
Article in English | WPRIM | ID: wpr-73247


Since Zika virus has been spreading rapidly in the Americas from 2015, the outbreak of Zika virus infection becomes a global health emergency because it can cause neurological complications and adverse fetal outcome including microcephaly. Here, we report clinical manifestations and virus isolation findings from a case of Zika virus infection imported from Brazil. The patient, 43-year-old Korean man, developed fever, myalgia, eyeball pain, and maculopapular rash, but not neurological manifestations. Zika virus was isolated from his semen, and reverse-transcriptase PCR was positive for the virus in the blood, urine, and saliva on the 7th day of the illness but was negative on the 21st day. He recovered spontaneously without any neurological complications. He is the first case of Zika virus infection in Korea imported from Brazil.

Adult , Brazil , Humans , Male , Microscopy, Electron, Transmission , RNA, Viral/analysis , Republic of Korea , Reverse Transcriptase Polymerase Chain Reaction , Saliva/virology , Semen/virology , Travel , Zika Virus/genetics , Zika Virus Infection/diagnosis
Article in English | WPRIM | ID: wpr-59850


BACKGROUND: During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits. METHODS: PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively. RESULTS: The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition. CONCLUSIONS: The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls.

Coronavirus Infections/diagnosis , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Nasopharynx/virology , Open Reading Frames/genetics , RNA, Viral/analysis , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction , Viral Envelope Proteins/genetics
Article in English | WPRIM | ID: wpr-59849


BACKGROUND: Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. METHODS: We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). RESULTS: While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both P<0.0001). CONCLUSIONS: The PK-DNase method is suitable for homogenizing sputum samples prior to RNA extraction.

Acetylcysteine/chemistry , Citrates/chemistry , Coronavirus Infections/diagnosis , Deoxyribonuclease I/metabolism , Endopeptidase K/metabolism , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction , Sputum/virology
Article in English | WPRIM | ID: wpr-225574


During the 2015 outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in Korea, 186 persons were infected, resulting in 38 fatalities. We isolated MERS-CoV from the oropharyngeal sample obtained from a patient of the outbreak. Cytopathic effects showing detachment and rounding of cells were observed in Vero cell cultures 3 days after inoculation of the sample. Spherical virus particles were observed by transmission electron microscopy. Full-length genome sequence of the virus isolate was obtained and phylogenetic analyses showed that it clustered with clade B of MERS-CoV.

Animals , Chlorocebus aethiops , Coronavirus Infections/diagnosis , Disease Outbreaks , Humans , Microscopy, Electron , Middle East Respiratory Syndrome Coronavirus/classification , Phylogeny , Polymerase Chain Reaction , RNA, Viral/analysis , Republic of Korea/epidemiology , Sequence Analysis, RNA , Vero Cells
Article in English | WPRIM | ID: wpr-56708


For two months between May and July 2015, a nationwide outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) occurred in Korea. On June 3, 2015, the Korean Society for Laboratory Medicine (KSLM) launched a MERS-CoV Laboratory Response Task Force (LR-TF) to facilitate clinical laboratories to set up the diagnosis of MERS-CoV infection. Based on the WHO interim recommendations, the Centers for Disease Control and Prevention of United States guidelines for MERS-CoV laboratory testing, and other available resources, the KSLM MERS-CoV LR-TF provided the first version of the laboratory practice guidelines for the molecular diagnosis of MERS-CoV to the clinical laboratories on June 12, 2015. The guidelines described here are an updated version that includes case definition, indications for testing, specimen type and protocols for specimen collection, specimen packing and transport, specimen handling and nucleic acid extraction, molecular detection of MERS-CoV, interpretation of results and reporting, and laboratory safety. The KSLM guidelines mainly focus on the molecular diagnosis of MERS-CoV, reflecting the unique situation in Korea and the state of knowledge at the time of publication.

Coronavirus Infections/diagnosis , Disease Outbreaks , Humans , Laboratories/standards , Middle East Respiratory Syndrome Coronavirus/genetics , Product Packaging/standards , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction , Republic of Korea/epidemiology , Societies, Scientific , Specimen Handling/standards
Article in English | WPRIM | ID: wpr-56704


BACKGROUND: The largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infection outside Middle East Asia in 2015 has necessitated the rapid expansion of laboratories that conduct MERS-CoV molecular testing in Korea, together with external quality assessment (EQA) to evaluate the assays used. METHODS: The EQA program consisted of two phases; self-validation and blind assessment. For the first EQA phase, in vitro transcribed upstream region of the envelope gene (upE) and the open reading frame (ORF)1a RNAs were used at a concentration of 1,000 copies/microL. The test panel for the second EQA phase consisted of RNA extracts from three samples, which were obtained from two MERS-CoV positive patients and one MERS-CoV negative patient. RESULTS: The first EQA phase results for 46 participants showed a linear relationship between the threshold cycle (CT) values of RNA materials and the logarithmic concentrations for both upE and ORF1a gene targets (R2=0.73 and 0.75, respectively). The mean CT value for each concentration was different depending on which commercial kit was used for the assay. Among the three commonly used kits, PowerChek MERS Real-Time PCR kit (KogeneBiotech, Korea) showed the lowest CT values at all concentrations of upE and most concentrations of ORF1a. The second EQA phase results for 47 participants were 100% correct for all tested samples. CONCLUSIONS: This EQA survey demonstrates that the MERS-CoV molecular testing performed in Korea during the 2015 outbreak is of robust capability. However, careful establishment and validation of a cut-off value are recommended to ensure good analytical sensitivity.

Coronavirus Infections/diagnosis , Disease Outbreaks , Humans , Middle East Respiratory Syndrome Coronavirus/genetics , Molecular Diagnostic Techniques/standards , Quality Assurance, Health Care , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction , Republic of Korea/epidemiology , Surveys and Questionnaires
Article in English | WPRIM | ID: wpr-56700


Rapid and accurate identification of an influenza outbreak is essential for patient care and treatment. We describe a next-generation sequencing (NGS)-based, unbiased deep sequencing method in clinical specimens to investigate an influenza outbreak. Nasopharyngeal swabs from patients were collected for molecular epidemiological analysis. Total RNA was sequenced by using the NGS technology as paired-end 250 bp reads. Total of 7 to 12 million reads were obtained. After mapping to the human reference genome, we analyzed the 3-4% of reads that originated from a non-human source. A BLAST search of the contigs reconstructed de novo revealed high sequence similarity with that of the pandemic H1N1 virus. In the phylogenetic analysis, the HA gene of our samples clustered closely with that of A/Senegal/VR785/2010(H1N1), A/Wisconsin/11/2013(H1N1), and A/Korea/01/2009(H1N1), and the NA gene of our samples clustered closely with A/Wisconsin/11/2013(H1N1). This study suggests that NGS-based unbiased sequencing can be effectively applied to investigate molecular characteristics of nosocomial influenza outbreak by using clinical specimens such as nasopharyngeal swabs.

Databases, Genetic , Genotype , High-Throughput Nucleotide Sequencing , Humans , Influenza A Virus, H1N1 Subtype/classification , Influenza, Human/diagnosis , Nasopharynx/virology , Nucleic Acid Amplification Techniques , Phylogeny , RNA, Viral/analysis , Sequence Analysis, RNA , Viral Proteins/genetics
Rev. Soc. Bras. Med. Trop ; 48(4): 468-470, July-Aug. 2015. tab
Article in English | LILACS | ID: lil-755971



Data on hepatitis E virus (HEV) in Brazil are limited. We analyzed 15 years of HEV surveillance data in a major clinical laboratory in São Paulo, Brazil.


The seroprevalence of HEV of 2,271 patients subjected to anti-HEV tests from 1998 to 2013 were analyzed.


HEV seroprevalence was 2.1%, and the anti-HEV IgM positivity rate was 4.9%. Six hepatitis E patients were identified.


HEV seroprevalence and detection rates appear to have increased in recent years. Hepatitis E should be investigated further and included in the differential diagnosis of hepatitis in Brazil.


Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Young Adult , Hepatitis E virus , Hepatitis E/epidemiology , Brazil/epidemiology , Hepatitis Antibodies/blood , Hepatitis E virus/genetics , Hepatitis E virus/immunology , Hepatitis E/diagnosis , Immunoglobulin G/blood , Immunoglobulin M/blood , Retrospective Studies , RNA, Viral/analysis , Seroepidemiologic Studies
Mem. Inst. Oswaldo Cruz ; 109(7): 912-917, 11/2014. tab, graf
Article in English | LILACS | ID: lil-728806


After the World Health Organization officially declared the end of the first pandemic of the XXI century in August 2010, the influenza A(H1N1)pdm09 virus has been disseminated in the human population. In spite of its sustained circulation, very little on phylogenetic data or oseltamivir (OST) resistance is available for the virus in equatorial regions of South America. In order to shed more light on this topic, we analysed the haemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09 positive samples collected during the pandemic period in the Pernambuco (PE), a northeastern Brazilian state. Complete HA sequences were compared and amino acid changes were related to clinical outcome. In addition, the H275Y substitution in NA, associated with OST resistance, was investigated by pyrosequencing. Samples from PE were grouped in phylogenetic clades 6 and 7, being clustered together with sequences from South and Southeast Brazil. The D222N/G HA gene mutation, associated with severity, was found in one deceased patient that was pregnant. Additionally, the HA mutation K308E, which appeared in Brazil in 2010 and was only detected worldwide the following year, was identified in samples from hospitalised cases. The resistance marker H275Y was not identified in samples tested. However, broader studies are needed to establish the real frequency of resistance in this Brazilian region.

Female , Humans , Pregnancy , Hemagglutinins/genetics , Influenza A Virus, H1N1 Subtype/genetics , Influenza, Human/epidemiology , Neuraminidase/genetics , Pandemics , Antiviral Agents/therapeutic use , Biomarkers/analysis , Brazil/epidemiology , Drug Resistance, Viral/physiology , Gene Frequency/genetics , Influenza A Virus, H1N1 Subtype/classification , Influenza A Virus, H1N1 Subtype/pathogenicity , Influenza, Human/virology , Mutation/genetics , Oseltamivir/therapeutic use , Phylogeny , RNA, Viral/analysis , Sequence Analysis, DNA/methods , Virulence , Virulence Factors/genetics